How are cardiac progenitor cells primed to differentiate into cardiomyocytes? This question has been the subject of numerous investigations over the past two decades. While, these experiments have identified several molecular players that are involved in signaling or transcription regulation pathways at one or more stages of differentiation, a comprehensive and quantitative picture of the cellular networks leading to cardiomyogenesis is yet to emerge. This is a primary goal of our proposal. We will begin with a legacy knowledge of the coarse-grained picture of initiation of stage-specific differentiation processes and conduct information-rich assays to obtain qualitative and quantitative knowledge of the players and signaling modules involved. The first set of assays will combine signaling module knowledge with introduction of module-specific inhibitors to get a first glimpse at which modules are activated or repressed at which stages of differentiation. This will be followed by phosphoproteomic analysis to identify specific phosphorylation cascades and provide us a more detailed picture of the signaling networks. We will expand this further using stage-specific gene- expression measurements and analyze the integrated sets of data to obtain more fine-grained pathways that lead to cardiomyogenesis. Most importantly, we will use spatio-temporal measurements of intracellular calcium to develop a quantitative model of the signaling networks that will help map input to response in activation of precursor cells towards cardiomyogenesis. This quantitative systems biology approach towards understanding one of the most important processes in regenerative medicine, that of cardiac formation from ESCs has the potential first to provide insights into the combinatorial complexity of signaling modules that operate as cells progress through the cardiomyogenic program. This will provide us vital information on specific triggers and activators needed to efficiently differentiate ESCs into cardiomyocytes. Second, we will develop both a parts list and a detailed network map of events at each stage of differentiation to build a systems biology perspective on differentiation. Third and most important, we will provide the quantitative framework for mapping input to response, i.e. phenotype in cardiomyogenesis. Finally, identification of ligands and molecules associated with our experiments will provide interesting therapeutic targets. The project brings together experts in stem cell biology, gene expression and quantitative systems biology in an exceptionally synergistic manner and will provide the community with invaluable data, models and hypotheses for biomedicine. Heart disease is the most common birth defect in humans. Successful completion of this proposal would greatly aid in our current understanding of heart development and disease and in the longer term highlight specific molecular pathways for future studies and thus, potential targets for future cardiac drug therapies.